结核分枝杆菌Rv0394c蛋白的免疫原性及其用于牛结核病诊断价值评价

Immunogenicity of Mycobacterium tuberculosis Rv0394c protein and its diagnosis potential for bovine tuberculosis

摘要:

为了评价结核分枝杆菌Rv0394c蛋白用于牛结核病诊断的潜力，利用原核表达系统对Rv0394c蛋白进行表达和纯化，通过小鼠和豚鼠模型评价其免疫原性，并分析其用于外周血IFN-γ释放实验的刺激效果。构建pColdI-Rv0394c原核表达质粒，并转化至大肠杆菌BL21(DE3)中诱导蛋白的表达，对蛋白进行纯化。评价纯化的重组蛋白免疫小鼠的免疫原性，通过灭活牛分枝杆菌致敏豚鼠皮试反应的大小评价蛋白作为皮肤变态反应原的诊断效果，并通过牛结核病外周血γ-干扰素(interferon-γ, IFN-γ)释放实验，评价其作为特异性刺激抗原的潜力。SDS-PAGE和Western blotting结果显示，成功获得可溶性的Rv0394c蛋白，且具有良好免疫反应性；小鼠实验显示Rv0394c蛋白刺激免疫小鼠脾脏淋巴细胞产生高水平特异性IFN-γ应答；豚鼠皮试结果显示Rv0394c蛋白可诱导致敏豚鼠产生较强的阳性反应；在牛结核病外周血IFN-γ释放实验中，Rv0394c蛋白可刺激结核阳性牛产生高水平的IFN-γ，与融合蛋白CFP10-ESAT6 (CE)混合使用，能够显著提高牛结核诊断灵敏度。Rv0394c蛋白是一种免疫优势抗原，具有作为皮试检测变态反应原、牛结核病外周血IFN-γ释放实验刺激原的潜力，为我国牛结核病新型诊断技术的开发提供了重要生物材料。

Abstract:

To evaluate the potential of Mycobacterium tuberculosis Rv0394c protein for use in the diagnosis of bovine tuberculosis, the Rv0394c protein was expressed and purified via a prokaryotic expression system. Its immunogenicity was assessed in mouse and guinea pig models, and its stimulatory effect in peripheral blood IFN-γ release assays was analyzed. The gene fragment of Rv0394c was introduced into the prokaryotic expression vector pColdI via homologous recombination to construct the recombinant plasmid pColdI-Rv0394c, which was transformed into Escherichia coli BL21(DE3) for induced expression. The purified recombinant protein was used to immunize mice to evaluate its immunogenicity. Its diagnostic efficacy as a skin hypersensitivity antigen was assessed in guinea pigs sensitized with inactivated Mycobacterium bovis. Additionally, its potential as a specific stimulating antigen was evaluated by the peripheral blood IFN-γ release assay for bovine tuberculosis. The Rv0394c fusion protein with good immunogenicity was successfully constructed and purified. Mouse experiments showed that Rv0394c stimulated splenic lymphocytes from immunized mice to produce high levels of specific IFN-γ. Skin test results demonstrated that Rv0394c elicited strong positive reactions in sensitized guinea pigs. In the bovine peripheral blood IFN-γ release assay, Rv0394c stimulated high levels of IFN-γ production in tuberculosis-positive cattle. When being used in combination with the fusion protein CFP10-ESAT6 (CE), Rv0394c significantly improved the diagnostic sensitivity for bovine tuberculosis. Rv0394c is an immunodominant antigen with the potential to serve as an allergen for skin test diagnosis and as a stimulating antigen for the peripheral blood IFN-γ release assay in bovine tuberculosis, which provides important biological materials for the development of novel diagnostic technologies for bovine tuberculosis.